Project Summary/Abstract Research. The goal of this K99/R00 project is to test the hypothesis that cytochromes P450 from Actinomycetales, a diverse subfamily of remarkable biocatalysts, may be used as a means to discover natural products with novel functionalities. We propose to (Aim 1) investigate the sequence?structure?function relationships of novel P450s from selected natural product biosynthetic pathways, (Aim 2) use a P450-focused genome mining approach to discover natural products that have undergone unique P450-catalyzed transformations, and (Aim 3) employ the newly identified P450s, and their homologues, as models for P450 enzymology and bioengineering. The multidisciplinary approach involves bioinformatics analysis, in vitro enzymology, protein X-ray crystallography, in vivo pathway engineering, (un)natural product isolation and structural determination. Superb preliminary data supports both the feasibility of the proposed mentored phase in a two-year time span, and a successful transition to an independent research program. The innovation of this application lies in leveraging the diversity of natural P450 variants from a proprietary Actinomycetales strain collection for discovering unprecedented chemistries that imbue natural products with new functionalities. The significance of this project is the advancement of P450 catalysis and enzymology and the discovery of novel natural products, laying the foundation for future treatments of human diseases. Candidate and Environment. Dr. Rudolf obtained graduate training in mechanistic enzymology and organic synthesis from Dr. C. Dale Poulter, a preeminent terpenoid biochemist, at the University of Utah. He then joined Dr. Ben Shen, a recognized leader in natural products discovery and biosynthesis, at The Scripps Research Institute (TSRI), and received postdoctoral training in actinomycetes genetics and natural products biosynthesis. His long-term career goals include establishing an independent, competitive research program that emphasizes using natural products as inspirations for the discovery and study of novel chemistries, enzymologies, and biologies. His immediate career goals include obtaining training in P450 enzymology and structural biology, and in utilizing genomics for the discovery of novel enzymes and natural products. Career development plan. This proposal was designed to ensure the realization of his career goals by providing intellectual, technical, and professional training during the mentored phase that will lead to an effective transition to an independent, tenure-track position. The primary mentor, Dr. Shen, and co-mentor, Dr. George Phillips, an expert in X-ray crystallography and heme proteins, were chosen for their scientific expertise and mentoring experience. An advisory committee, including Drs. Bradley Moore, Wilfred van der Donk, Sean Brady, and Michael Smanski, all experts in natural products, was explicitly assembled to oversee both the science and career development aspects of this proposal. The research training and excellent mentoring will be supplemented with numerous opportunities for scientific and professional development available at TSRI.